Methods for uniform metal impregnation into a nanoporous material

ABSTRACT

The methods, systems  400  and apparatus disclosed herein concern metal  150  impregnated porous substrates  110, 210.  Certain embodiments of the invention concern methods for producing metal-coated porous silicon substrates  110, 210  that exhibit greatly improved uniformity and depth of penetration of metal  150  deposition. The increased uniformity and depth allow improved and more reproducible Raman detection of analytes. In exemplary embodiments of the invention, the methods may comprise oxidation of porous silicon  110,  immersion in a metal salt solution  130,  drying and thermal decomposition of the metal salt  140  to form a metal deposit  150.  In other exemplary embodiments of the invention, the methods may comprise microfluidic impregnation of porous silicon substrates  210  with one or more metal salt solutions  130.  Other embodiments of the invention concern apparatus and/or systems  400  for Raman detection of analytes, comprising metal-coated porous silicon substrates  110, 210  prepared by the disclosed methods.

FIELD

[0001] The present methods and apparatus relate to the field of metal150 impregnation into nanoporous materials 110, 210. More particularly,certain embodiments of the invention concern methods of producingmetal-coated porous silicon 110, 210.

BACKGROUND

[0002] The sensitive and accurate detection and/or identification ofsingle molecules from biological and other samples has proven to be anelusive goal, with widespread potential uses in medical diagnostics,pathology, toxicology, biological warfare, environmental sampling,chemical analysis, forensics and numerous other fields. Attempts havebeen made to use Raman spectroscopy and/or surface plasmon resonance toachieve this goal. When light passes through a tangible medium, acertain amount becomes diverted from its original direction, aphenomenon known as Raman scattering. Some of the scattered light alsodiffers in frequency from the original excitatory light, due to theabsorption of light and excitation of electrons to a higher energystate, followed by light emission at a different wavelength. Thewavelengths of the Raman emission spectrum are characteristic of thechemical composition and structure of the light absorbing molecules in asample, while the intensity of light scattering is dependent on theconcentration of molecules in the sample.

[0003] The probability of Raman interaction occurring between anexcitatory light beam and an individual molecule in a sample is verylow, resulting in a low sensitivity and limited applicability of Ramananalysis. It has been observed that molecules near roughened silversurfaces show enhanced Raman scattering of as much as six to sevenorders of magnitude. This surface enhanced Raman spectroscopy (SERS)effect is related to the phenomenon of plasmon resonance, wherein metalnanoparticles exhibit a pronounced optical resonance in response toincident electromagnetic radiation, due to the collective coupling ofconduction electrons in the metal. In essence, nanoparticles of gold,silver, copper and certain other metals can function as miniature“antenna” to enhance the localized effects of electromagnetic radiation.Molecules located in the vicinity of such particles exhibit a muchgreater sensitivity for Raman spectroscopic analysis.

[0004] Attempts have been made to exploit SERS for molecular detectionand analysis, typically by coating metal nanoparticles or fabricatingrough metal films on the surface of a substrate and then applying asample to the metal-coated surface. However, the number of metalparticles that can be deposited on a planar surface is limited,producing a relatively low enhancement factor for SERS and related Ramantechniques utilizing such surfaces. A need exists for methods ofproducing SERS-active substrates with uniform, high densities ofRaman-active metal.

[0005] Metal impregnated silicon substrates have been proposed ascomponents of various electrical devices, such as field emissionelectron sources and light emitting diodes. The efficiency of suchdevices is limited by a lack of uniformity of electrical contacts,resulting from non-homogeneous metal impregnation. A need exists formethods of producing materials with homogeneous metal impregnation forhigh efficiency electrical devices.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the disclosedembodiments of the invention. The embodiments of the invention may bebetter understood by reference to one or more of these drawings incombination with the detailed description of specific embodimentspresented herein.

[0007]FIG. 1 illustrates an exemplary method for producing ametal-coated porous silicon substrate 110 comprising thermaldecomposition of a metal salt solution 130. FIG. 1A shows a poroussilicon substrate 110. FIG. 1B illustrates silicon oxidation, forexample by plasma oxidation, to form a layer of silicon dioxide 120.FIG. 1C shows immersion of the oxidized porous silicon 110 in a metalsalt solution 130, such as a silver nitrate solution 130. FIG. 1Dillustrates removal of excess metal salt solution 130. FIG. 1E showsdrying of the solution 130 to form a thin layer of dry metal salt 140 onthe porous silicon substrate 110. FIG. 1F illustrates thermaldecomposition of the dry metal salt 140 to form a uniform layer of metal150 coating the porous silicon substrate 110.

[0008]FIG. 2 illustrates another exemplary method for producing ametal-coated porous silicon substrate 210 comprising microfluidicimpregnation.

[0009]FIG. 3 illustrates an alternative embodiment of the invention fordelivering different metal plating solutions to a porous siliconsubstrate 210.

[0010]FIG. 4 shows an exemplary system 400 for detecting various targetmolecules using a metal-coated porous silicon substrate 210 and Ramandetection.

[0011]FIG. 5 illustrates the uniform deposition of an exemplary metal150 (silver) on a porous silicon substrate 110 using a thermaldecomposition method.

[0012]FIG. 6 shows the surface-enhanced Raman spectrum for an exemplaryanalyte, rhodamine 6G (R6G) dye molecules, obtained with aplasma-oxidized, dip and decomposed (PODD) porous silicon substrate 110uniformly coated with silver 150. The PODD substrate 110 was prepared bythe method of FIG. 1. A solution of 114 μM (micromolar) R6G moleculeswas subjected to SERS (surface enhanced Raman spectroscopy) usingexcitation at 785 nm (nanometers). FIG. 6 shows the SERS emissionspectra obtained with PODD silver-coated substrates 110 of differentporosities. The various spectra were obtained at average porosities, inorder from the lowest trace to the highest trace, of 52%, 55%, 65%, 70%and 77%.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0013] The following detailed description contains numerous specificdetails in order to provide a more thorough understanding of thedisclosed embodiments of the invention. However, it will be apparent tothose skilled in the art that the embodiments of the invention may bepracticed without these specific details. In other instances, devices,methods, procedures, and individual components that are well known inthe art have not been described in detail herein.

[0014] Definitions

[0015] As used herein, “a” or “an” may mean one or more than one of anitem.

[0016] As used herein, the terms “analyte” and “target” refer to anyatom, chemical, molecule, compound, composition or aggregate of interestfor detection and/or identification. Non-limiting examples of analytesinclude an amino acid, peptide, polypeptide, protein, glycoprotein,lipoprotein, nucleoside, nucleotide, oligonucleotide, nucleic acid,sugar, carbohydrate, oligosaccharide, polysaccharide, fatty acid, lipid,hormone, metabolite, cytokine, chemokine, receptor, neurotransmitter,antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor,drug, pharmaceutical, nutrient, prion, toxin, poison, explosive,pesticide, chemical warfare agent, biohazardous agent, radioisotope,vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic,amphetamine, barbiturate, hallucinogen, waste product and/orcontaminant. In certain embodiments of the invention, one or moreanalytes may be labeled with one or more Raman labels.

[0017] As used herein, the term “nanocrystalline silicon” refers tosilicon that comprises nanometer-scale silicon crystals, typically inthe size range from 1 to 100 nanometers (nm). “Porous silicon” 110, 210refers to silicon that has been etched or otherwise treated to form aporous structure 110, 210.

[0018] As used herein, “operably coupled” means that there is afunctional interaction between two or more units of an apparatus and/orsystem. For example, a Raman detector 410 may be “operably coupled” to acomputer if the computer can obtain, process, store and/or transmit dataon Raman signals detected by the detector 410.

[0019] Porous Substrates

[0020] Certain embodiments of the invention concern methods for coatingporous substrates 110, 210 with a uniform layer of one or more metals150, such as Raman active metals 150. Although in particular embodimentsof the invention the porous substrates 110, 210 disclosed herein areporous silicon substrates 110, 210, those embodiments are not limiting.Any porous substrate 110, 210 that is resistant to the application ofheat may be used in the disclosed methods, systems 400 and/or apparatus.In certain embodiments, application of heat to about 300° C., 400° C.,500° C., 600° C., 700° C., 800° C., 900° C. or 1,000° C. iscontemplated. In some embodiments of the invention, the porous substrate110, 210 may be rigid. A variety of porous substrates 110, 210 areknown, including but not limited to porous silicon, porous polysilicon,porous metal grids and porous aluminum. Exemplary methods of makingporous substrates 110, 210 are disclosed in further detail below.

[0021] Porous polysilicon substrates 110, 210 may be made by knowntechniques (e.g., U.S. Pat. Nos. 6,249,080 and 6,478,974). For example,a layer of porous polysilicon 110, 210 may be formed on top of asemiconductor substrate by the use of low pressure chemical vapordeposition (LPCVD). The LPCVD conditions may include, for example, apressure of about 20 pascal, a temperature of about 640° C. and a silanegas flow of about 600 sccm (standard cubic centimeters) (U.S. Pat. No.6,249,080). A polysilicon layer may be etched, for example usingelectrochemical anodization with HF (hydrofluoric acid) or chemicaletching with nitric acid and hydrofluoric acid, to make it porous (U.S.Pat. No. 6,478,974). Typically, porous polysilicon 110, 210 layersformed by such techniques are limited in thickness to about 1 μm(micrometer) or less. In contrast, porous silicon 110, 210 can be etchedthroughout the thickness of the bulk silicon wafer, which has a typicalthickness of about 500 μm.

[0022] Porous aluminum substrates 110, 210 may also be made by knowntechniques (e.g., Cai et al., Nanotechnology 13:627, 2002; Varghese etal., J. Mater. Res. 17:1162-1171, 2002). For example, nanoporousaluminum oxide thin films 110, 210 may be fabricated on silicon orsilicon dioxide 120 using an electrochemical-assisted self-assemblyprocess (Cai et al., 2002). The porous aluminum film 110, 210 may bethermally annealed to improve its uniformity (Cai et al., 2002).Alternatively, a thin layer of solid aluminum may be electrochemicallyanodized in dilute solutions of oxalic acid and/or sulfuric acid tocreate a nanoporous alumina film 110, 210 (Varghese et al., 2002). Theexamples disclosed herein are not limiting and any known type of heatresistant porous substrate 110, 210 may be used. Such porous substrates110, 210 may be uniformly impregnated with one or more metals 150, suchas silver, using the methods disclosed herein.

[0023] Nanocrystalline Porous Silicon

[0024] Nanocrystalline Silicon

[0025] Certain embodiments of the invention concern systems 400 and/orapparatus comprising one or more layers of nanocrystalline silicon.Various methods for producing nanocrystalline silicon are known (e.g.,Petrova-Koch et al., “Rapid-thermal-oxidized porous silicon—the superiorphotoluminescent Si,” Appl. Phys. Lett. 61:943, 1992; Edelberg, et al.,“Visible luminescence from nanocrystalline silicon films produced byplasma enhanced chemical vapor deposition,” Appl. Phys. Lett.,68:1415-1417, 1996; Schoenfeld, et al., “Formation of Si quantum dots innanocrystalline silicon,” Proc. 7th Int. Conf. on ModulatedSemiconductor Structures, Madrid, pp. 605-608, 1995; Zhao, et al.,“Nanocrystalline Si: a material constructed by Si quantum dots,” 1stInt. Conf. on Low Dimensional Structures and Devices, Singapore, pp.467-471, 1995; Lutzen et al., Structural characteristics of ultrathinnanocrystalline silicon films formed by annealing amorphous silicon, J.Vac. Sci. Technology B 16:2802-05, 1998; U.S. Pat. Nos. 5,770,022;5,994,164; 6,268,041; 6,294,442; 6,300,193). The methods, systems 400and apparatus disclosed herein are not limited by the method ofproducing nanocrystalline silicon and any known method may be used.

[0026] Non-limiting exemplary methods for producing nanocrystallinesilicon include silicon (Si) implantation into a silicon rich oxide andannealing; solid phase crystallization with metal nucleation catalysts;chemical vapor deposition; PECVD (plasma enhanced chemical vapordeposition); gas evaporation; gas phase pyrolysis; gas phasephotopyrolysis; electrochemical etching; plasma decomposition of silanesand polysilanes; high pressure liquid phase reduction-oxidationreactions; rapid annealing of amorphous silicon layers; depositing anamorphous silicon layer using LPCVD (low pressure chemical vapordeposition) followed by RTA (rapid thermal anneal) cycles; plasmaelectric arc deposition using a silicon anode and laser ablation ofsilicon (U.S. Pat. Nos. 5,770,022; 5,994,164; 6,268,041; 6,294,442;6,300,193). Depending on the process, Si crystals of anywhere from 1 to100 nm or more in size may be formed as a thin layer on a chip, aseparate layer and/or as aggregated crystals. In certain embodiments ofthe invention, a thin layer comprising nanocrystalline silicon attachedto a substrate layer may be used.

[0027] In various embodiments of the invention, it is contemplated thatnanocrystalline silicon may be used to form a porous silicon substrate110, 210. However, the embodiments are not limited to as to thecomposition of the starting material, and in alternative embodiments ofthe invention it is contemplated that other materials may be utilized,provided that the material is capable of forming a porous substrate 110,210 that can be coated with a metal 150, as exemplified in FIG. 1.

[0028] In certain embodiments of the invention, the size and/or shape ofsilicon crystals and/or pore size in porous silicon 110, 210 may beselected to be within predetermined limits, for example, in order tooptimize the plasmon resonant frequency of metal-coated porous silicon110, 210 (see, e.g., U.S. Pat. No. 6,344,272). Techniques forcontrolling the size of nanoscale silicon crystals are known (e.g., U.S.Pat. Nos. 5,994,164 and 6,294,442). The plasmon resonant frequency mayalso be adjusted by controlling the thickness and/or composition of themetal layer 150 coating the porous silicon 110, 210 (U.S. Pat. No.6,344,272).

[0029] Porous Silicon

[0030] Certain embodiments of the invention concern systems 400 and/orapparatus comprising a metal-coated porous substrate 110, 210. Invarious embodiments, the substrate may comprise nanocrystalline poroussilicon 110, 210. The substrate is not limited to pure silicon, but mayalso comprise silicon nitride, silicon oxide, silicon dioxide 120,germanium and/or other materials known for chip manufacture. Other minoramounts of material may also be present, such as dopants. Porous silicon110, 210 has a large surface area of up to 783 m²/cm³, providing a verylarge surface for applications such as surface enhanced Ramanspectroscopy techniques.

[0031] Porous silicon 110, 210 was discovered in the late 1950's byelectropolishing silicon in dilute hydrofluoric acid solutions. As isknown in the art, porous silicon 110, 210 may be produced by etching asilicon substrate with dilute hydrofluoric acid (HF) in anelectrochemical cell. In certain cases, silicon may be initially etchedin HF at low current densities. After the initial pores are formed, thesilicon may be removed from the electrochemical cell and etched in verydilute HF to widen the pores formed in the electrochemical cell. Thecomposition of the porous silicon substrate 110, 210 will also affectpore size, depending on whether or not the silicon is doped, the type ofdopant and the degree of doping. The effect of doping on silicon poresize is known in the art. For embodiments of the invention involvingdetection and/or identification of large biomolecules, a pore size ofabout 2 nm to 100 or 200 nm may be selected. The orientation of pores inporous silicon 110, 210 may also be selected in particular embodimentsof the invention. For example, an etched 1,0,0 crystal structure willhave pores oriented perpendicular to the crystals, while 1,1,1 or 1,1,0crystal structures will have pores oriented diagonally along the crystalaxis. The effect of crystal structure on pore orientation is also knownin the art. Crystal composition and porosity may also be regulated tochange the optical properties of the porous silicon 110, 210. Suchproperties may be changed, for example, to enhance Raman signals anddecrease background noise and/or to optimize the characteristics oflight emitting diodes or field emission electron sources incorporatingmetal-coated porous silicon 110, 210. The optical properties of poroussilicon 110, 210 are known in the art (e.g., Cullis et al., J. Appl.Phys. 82:909-965, 1997; Collins et al., Physics Today 50:24-31, 1997).

[0032] In a non-limiting example of a method for producing a poroussilicon substrate 110, 210, a silicon wafer may be placed inside anelectrochemical cell comprising an inert material, such as Teflon®. Thewafer is connected to the positive pole of a constant current source,forming the anode of the electrochemical cell. The negative pole of theconstant current source is connected to a cathode, such as a platinumelectrode. The electrochemical cell may be filled with a diluteelectrolyte solution of HF in ethanol. Alternatively, HF may bedissolved in other alcohols and/or surfactants known in the art, such aspentane or hexane. In certain embodiments of the invention, a computermay be operably coupled to a constant current source to regulate thecurrent, voltage and/or time of electrochemical etching. The siliconwafer exposed to HF electrolyte in the electrochemical cell becomesetched to form a porous silicon substrate 110, 210. As is known in theart, the thickness of the porous silicon layer 110, 210 and the degreeof porosity of the silicon may be controlled by regulating the timeand/or current density of anodization and the concentration of HF in theelectrolyte solution (e.g., U.S. Pat. No. 6,358,815).

[0033] In various embodiments of the invention, portions of the siliconwafer may be protected from HF etching by coating with any known resistcompound, such as polymethyl-methacrylate. Lithography methods, such asphotolithography, of use for exposing selected portions of a siliconwafer to HF etching are well known in the art. Selective etching may beof use to control the size and shape of a porous Si chamber 110, 210 tobe used for Raman spectroscopy or for various electrical devices. Incertain embodiments of the invention, a porous silicon chamber 110, 210of about 1 μm (micrometer) in diameter may be used. In other embodimentsof the invention, a trench or channel of porous silicon 110, 210 ofabout 1 μm in width may be used. The size of the porous silicon chamber110, 210 is not limiting, and it is contemplated that any size or shapeof porous silicon chamber 110, 210 may be used. A 1 μm chamber size maybe of use, for example, with an excitatory laser 410 that emits a lightbeam of about 1 μm in size.

[0034] The exemplary method above is not limiting for producing poroussilicon substrates 110, 210 and it is contemplated that any method knownin the art may be used. Non-limiting examples of methods for makingporous silicon substrates 110, 210 include anodic etching of siliconwafers and depositing a silicon/oxygen containing material followed bycontrolled annealing (e.g., Canham, “Silicon quantum wire arrayfabrication by electrochemical and chemical dissolution of wafers,”Appl. Phys. Lett. 57:1046, 1990; U.S. Pat. Nos. 5,561,304; 6,153,489;6,171,945; 6,322,895; 6,358,613; 6,358,815; 6,359,276). In variousembodiments of the invention, the porous silicon layer 110, 210 may beattached to one or more supporting layers, such as bulk silicon, quartz,glass and/or plastic. In certain embodiments, an etch stop layer, suchas silicon nitride, may be used to control the depth of etching. Theporous silicon layer 110, 210 may be incorporated into a semiconductorchip, using known methods of chip manufacture. In certain embodiments ofthe invention, a metal-coated porous silicon 110, 210 chamber may bedesigned as part of an integral chip, connected to various channels,microchannels, nanochannels, microfluidic channels, reaction chambers,solvent reservoirs 220, waste reservoirs 230, etc. In alternativeembodiments, a metal-coated porous silicon 110, 210 chamber may be cutout of a silicon wafer and incorporated into a chip and/or other device.

[0035] In certain alternative embodiments of the invention, it iscontemplated that additional modifications to the porous siliconsubstrate 110, 210 may be made, either before or after metal 150coating. For example, after etching a porous silicon substrate 110, 210may be oxidized, using methods known in the art, to silicon oxide and/orsilicon dioxide 120. Oxidation may be used, for example, to increase themechanical strength and stability of the porous silicon substrate 110,210 and/or to prevent spontaneous immersion plating of porous silicon110, 210, which can lead to pore blockage of nanoscale channels.Alternatively, the metal-coated porous silicon substrate 110, 210 may besubjected to further etching to remove the silicon material, leaving ametal 150 shell that may be left hollow or may be filled with othermaterials, such as one or more additional metals 150.

[0036] Metal Coating of Porous Substrates

[0037] Porous substrates 110, 210, such as porous silicon 110, 210, maybe coated with a metal 150, such as a Raman active metal 150. ExemplaryRaman active metals 150 include, but are not limited to gold, silver,platinum, copper and aluminum. Known methods of metal 150 coatinginclude electroplating; cathodic electromigration; evaporation andsputtering of metals 150; using seed crystals to catalyze plating (i.e.using a copper/nickel seed to plate gold); ion implantation; diffusion;or any other method known in the art for plating thin metal layers 150on porous substrates 110, 210. (See, e.g., Lopez and Fauchet, “Erbiumemission from porous silicon one-dimensional photonic band gapstructures,” Appl. Phys. Lett. 77:3704-6, 2000; U.S. Pat. Nos.5,561,304; 6,171,945; 6,359,276.) Another non-limiting example of metal150 coating comprises electroless plating (e.g., Gole et al., “Patternedmetallization of porous silicon from electroless solution for directelectrical contact,” J. Electrochem. Soc. 147:3785, 2000). Thecomposition and/or thickness of the metal layer 150 may be controlled tooptimize optical and/or electrical characteristics of the metal-coatedporous substrates 110, 210.

[0038] Arsenic-anodized porous silicon 110, 210 is known to function asa moderate reducing agent for metal ions, thereby initiating spontaneousimmersion plating of metal 150 on the top surface of the porous area110, 210 and closing the pore openings. Thus, using standard methods ofmetal 150 impregnation, it is difficult to obtain a uniform metal 150depth profile while maintaining an open porous surface 110, 210. Thereis a trade-off between the unblocked pores and metal 150 penetrationdepth, which can be explained as follows. High concentrations of metalion are needed to obtain a better metal 150 depth profile. However,exposure to high concentrations of metal salt solutions 130 close thepores due to the thick metal film 150 deposition from the spontaneousimmersion plating reaction. To maintain an open pore, the concentrationof metal ion in solution 130 needs to be lower. However, this causespoorer penetration depth, as well as reducing the amount of metal 150deposited. This problem is resolved by the methods disclosed herein,which allow a more uniform metal 150 deposition without pore clogging.

[0039] Metal Coating by Thermal Decomposition of a Metal Salt

[0040] As illustrated in FIG. 1, in particular embodiments of theinvention a porous silicon substrate 110 may be uniformly coated with ametal 150, such as a Raman sensitive metal 150, by a method comprisingthermal decomposition of a metal salt layer 140. In particularembodiments of the invention, the metal 150 is silver. A porous siliconsubstrate 110 (FIG. 1A) may be obtained, for example, as disclosedabove. To prevent premature metal 150 deposition and pore blocking, thesurface layer of silicon may be oxidized to silicon dioxide 120 (FIG.1B), for example by chemical oxidation or plasma oxidation. Oxidationprevents spontaneous immersion plating by stabilizing the porous silicon110 surface. In the absence of oxidation, positively charged silvercations can engage in a redox reaction with unoxidized silicon,resulting in spontaneous silver metal 150 deposition.

[0041] Following oxidation, the porous silicon substrate 110 is wet witha metal salt solution 130, such as a 1 M solution of silver nitrate(AgNO₃) (FIG. 1C). In a non-limiting example, the oxidized poroussilicon substrate 110 is dipped into a silver nitrate solution 130 for20 minutes, until the pores are completely wet with the silver nitratesolution 130. Excess metal salt solution 130 is removed, for example, bynitrogen gun drying (FIG. 1D). The solution 130 remaining in the poresmay be dried, for example, by heating to 100° C. for 20 min. At thispoint, the solvent has evaporated and a thin layer of dry silver nitratesalt 140 is deposited on the surface of the porous silicon 110. The drysalt 140 may be thermally decomposed (FIG. 1F), for example by heatingto 500° C. for 30 min in an ambient pressure furnace. The reaction ofEquation 1 occurs spontaneously at temperatures above 573° K. (about300° C.). The nitrate ion is converted to gaseous nitrogen dioxideaccording to Equation 1, resulting in deposition of a uniform layer ofmetallic silver 150 coating the porous silicon substrate 110 (FIG. 1F).Although nitrogen dioxide has been used as a photoetching agent, underthe conditions of the disclosed method it does not appear to etch thesilicon dioxide layer 120.

AgNO₃→Ag(s)+NO₂(gas)+½O₂(gas)   (1)

[0042] The thickness of the deposited metal layer 150 may be controlled,for example, by varying the concentration of the metal salt solution130. Depending on the thickness of metal layer 150 to be deposited, thesalt solution 130 concentration can vary between a wide range, of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0,2.5, 3.0, 3.5, 4.0, 4.5 to 5.0 M (molar). Although the exemplary methodutilizes a silver solution 130, the embodiments of the invention are notlimited to depositing silver 150 but may encompass any known metal 150,including but not limited to Raman active metals 150 such as gold,copper, platinum, aluminum, etc. The methods are also not limited as tothe type of salt used. In certain embodiments of the invention, theanionic species used to form the metal salt may be one that is convertedto a gaseous species and driven off during the thermal decompositionprocess, such as nitrate or sulfate ion. However, in alternativeembodiments any anionic species without limitation may be used.

[0043] Metal Coating by Microfluidic Impregnation

[0044] In alternative embodiments of the invention illustrated in FIG.2, a porous membrane 210, such as a porous silicon membrane 210, may becoated with metal 150 using microfluidic impregnation. In an exemplarymethod, a porous silicon membrane 210 may be obtained as disclosedabove. The porous silicon layer 210 may be electropolished and suspendedin a solution. The electropolished membrane 210 may be inserted into amicrofluidic pathway between one or more solvent reservoirs 220 and awaste reservoir 230 that are connected through cross-paths 240. Suchmicrofluidic pathways may be produced by any method known in the art,such as micromolding with PDMS (polydimethyl siloxane), standardlithography techniques or photolithography and etching of various chipmaterials (e.g., Duffy et al., Anal. Chem. 70:4974-84, 1998). The poroussilicon membrane 210 may be incorporated into any type of microfluidicsystem. In certain embodiments of the invention, microfluidic systemsincorporating porous silicon membranes 210 may be of use for a widevariety of applications relating to analysis and/or separation ofpolymer molecules, including but not limited to proteins and nucleicacids. Methods for micro and/or nanoscale manufacturing are known in theart, as discussed in more detail below.

[0045] A metal salt solution 130, such as a silver nitrate solution 130,may be introduced through the solvent reservoir 220 and allowed to flowthrough the porous silicon membrane 210 to a waste reservoir 230. Aspontaneous reaction will occur, as indicated in Equation 2.

Ag⁺(aq.)+Si(surface)+2H₂O(liquid)→Ag(solid)+H₂(gas)+SiO₂(surface)+2H⁺  (2)

[0046] As disclosed in Equation 2, an aqueous metal solution 130 reactsspontaneously with a porous silicon surface 210 in a redox reaction,producing a deposited metal 150 coating on the porous silicon 210. Thethickness of the metal 150 coating may be controlled by the metal saltconcentration of the solution 130, the rate of flow through themicrofluidic pathway, the temperature, and/or the length of time thatthe solution 130 is allowed to flow through the membrane 210. Techniquesfor controlling such metal 150 plating reactions are known in the art.

[0047] The method is not limited to silver solutions 130, but may alsobe performed with solutions 130 of other metal salts, including but notlimited to Raman active metals 150 such as gold, platinum, aluminum,copper, etc. In other alternative embodiments of the invention, theporous silicon membrane 210 may be coated with two or more differentmetals 150, using multiple solvent reservoirs 220 containing differentmetal plating solutions 130 (FIG. 3). In certain embodiments of theinvention, one or more reservoirs 220 may contain a wash solution toremove excess metal plating solution 130. Coating with multiple metals150 may be used to manipulate the electrical, optical and/or Ramansurface characteristics of the metal-coated porous silicon membrane 210,such as the degree of surface enhancement of the Raman signal, thedistance from the surface 210 at which resonance occurs, the range ofwavelengths of Raman resonance, etc.

[0048] The disclosed methods result in the production of a metal-coatedporous silicon membrane 210 integrated into a microfluidic pathway. Suchan integrated microchip may be directly incorporated into a Ramandetection system 400 as exemplified in FIG. 4. One or more samplessuspected of containing target molecules may be loaded intocorresponding solvent reservoirs 220. Samples may be channeled throughthe microfluidic pathway to enter the metal-coated membrane 210. Once inthe membrane 210, the target molecule may be excited by an excitatorylight source 410, such as a laser 410. An emitted Raman signal may bedetected by a Raman detector 420, as discussed in more detail below.Once analyzed, samples may be removed into a waste reservoir 230, themembrane 210 washed and the next sample analyzed. The Raman detectionsystem 400 may incorporate various components known in the art, such asRaman detectors 420 and excitatory light sources 410, or may comprisecustom components designed to be fully integrated into the system 400 tooptimize Raman detection of analytes.

[0049] Micro-Electro-Mechanical Systems (MEMS)

[0050] In some embodiments of the invention, a metal-coated poroussilicon substrate 110, 210 may be incorporated into a larger apparatusand/or system 400. In certain embodiments, the substrate 110, 210 may beincorporated into a micro-electro-mechanical system (MEMS) 400. MEMS areintegrated systems 400 comprising mechanical elements, sensors,actuators, and electronics. All of those components may be manufacturedby known microfabrication techniques on a common chip, comprising asilicon-based or equivalent substrate (e.g., Voldman et al., Ann. Rev.Biomed. Eng. 1:401-425, 1999). The sensor components of MEMS may be usedto measure mechanical, thermal, biological, chemical, optical and/ormagnetic phenomena. The electronics may process the information from thesensors and control actuator components such pumps, valves, heaters,coolers, filters, etc. thereby controlling the function of the MEMS.

[0051] The electronic components of MEMS may be fabricated usingintegrated circuit (IC) processes (e.g., CMOS, Bipolar, or BICMOSprocesses). They may be patterned using photolithographic and etchingmethods known for computer chip manufacture. The micromechanicalcomponents may be fabricated using compatible “micromachining” processesthat selectively etch away parts of the silicon wafer or add newstructural layers to form the mechanical and/or electromechanicalcomponents.

[0052] Basic techniques in MEMS manufacture include depositing thinfilms of material on a substrate, applying a patterned mask on top ofthe films by photolithographic imaging or other known lithographicmethods, and selectively etching the films. A thin film may have athickness in the range of a few nanometers to 100 micrometers.Deposition techniques of use may include chemical procedures such aschemical vapor deposition (CVD), electrodeposition, epitaxy and thermaloxidation and physical procedures like physical vapor deposition (PVD)and casting. Methods for manufacture of nanoelectromechanical systemsmay be used for certain embodiments of the invention. (See, e.g.,Craighead, Science 290:1532-36, 2000.)

[0053] In some embodiments of the invention, metal-coated porous siliconsubstrates 110, 210 may be connected to various fluid filledcompartments, such as microfluidic channels, nanochannels and/ormicrochannels. These and other components of the apparatus may be formedas a single unit, for example in the form of a chip as known insemiconductor chips and/or microcapillary or microfluidic chips.Alternatively, the metal-coated porous silicon substrate 110, 210 may beremoved from a silicon wafer and attached to other components of anapparatus. Any materials known for use in such chips may be used in thedisclosed apparatus, including silicon, silicon dioxide 120, siliconnitride, polydimethyl siloxane (PDMS), polymethylmethacrylate (PMMA),plastic, glass, quartz, etc.

[0054] Techniques for batch fabrication of chips are well known in thefields of computer chip manufacture and/or microcapillary chipmanufacture. Such chips may be manufactured by any method known in theart, such as by photolithography and etching, laser ablation, injectionmolding, casting, molecular beam epitaxy, dip-pen nanolithography,chemical vapor deposition (CVD) fabrication, electron beam or focusedion beam technology or imprinting techniques. Non-limiting examplesinclude conventional molding with a flowable, optically clear materialsuch as plastic or glass; photolithography and dry etching of silicondioxide 120; electron beam lithography using polymethylmethacrylateresist to pattern an aluminum mask on a silicon dioxide 120 substrate,followed by reactive ion etching. Known methods for manufacture ofnanoelectromechanical systems may be used for certain embodiments of theinvention. (See, e.g., Craighead, Science 290:1532-36, 2000.) Variousforms of microfabricated chips are commercially available from, e.g.,Caliper Technologies Inc. (Mountain View, Calif.) and ACLARA BioSciencesInc. (Mountain View, Calif.).

[0055] In certain embodiments of the invention, part or all of theapparatus may be selected to be transparent to electromagnetic radiationat the excitation and emission frequencies used for Raman spectroscopy,such as glass, silicon, quartz or any other optically clear material.For fluid-filled compartments that may be exposed to variousbiomolecules, such as proteins, peptides, nucleic acids, nucleotides andthe like, the surfaces exposed to such molecules may be modified bycoating, for example to transform a surface from a hydrophobic to ahydrophilic surface and/or to decrease adsorption of molecules to asurface. Surface modification of common chip materials such as glass,silicon, quartz and/or PDMS is known in the art (e.g., U.S. Pat. No.6,263,286). Such modifications may include, but are not limited to,coating with commercially available capillary coatings (Supelco,Bellafonte, Pa.), silanes with various functional groups such aspolyethyleneoxide or acrylamide, or any other coating known in the art.

[0056] Raman Spectroscopy

[0057] In certain embodiments of the invention, the disclosed methods,systems 400 and apparatus are of use for the detection and/oridentification of analytes by surface enhanced Raman spectroscopy(SERS), surface enhanced resonance Raman spectroscopy (SERRS) and/orcoherent anti-Stokes Raman spectroscopy (CARS) detection. Compared toexisting techniques, the disclosed methods, systems 400 and apparatusprovide SERS active substrates with increased and more uniform metal 150density and greater depth of field of SERS enhancement, allowing moreefficient Raman detection and/or identification of analytes.

[0058] Previous methods for SERS detection of various analytes have usedcolloidal metal 150 particles, such as aggregated silver 150nanoparticles, that were typically coated onto a substrate and/orsupport (e.g., U.S. Pat. Nos. 5,306,403; 6,149,868; 6,174,677;6,376,177). While such arrangements occasionally allow SERS detectionwith as much as 10⁶ to 10⁸ increased sensitivity, they are not capableof single molecule detection of small analytes such as nucleotides, asdisclosed herein. Enhanced sensitivity of Raman detection is apparentlynot uniform within a colloidal particle aggregate, but rather depends onthe presence of “hot spots.” The physical structure of such hot spots,the range of distances from the metal 150 nanoparticles at whichenhanced sensitivity occurs, and the spatial relationships betweenaggregated nanoparticles and analytes that allow enhanced sensitivityhave not been characterized. Further, aggregated metal 150 nanoparticlesare inherently unstable in solution, with adverse effects on thereproducibility of single molecule detection. The present methods,systems 400 and apparatus provide a stable microenvironment for SERSdetection in which the physical conformation and density of theRaman-active metal 150 porous substrate 110, 210 may be preciselycontrolled, allowing reproducible, sensitive and accurate detection ofanalytes in solution.

[0059] Raman Detectors

[0060] In some embodiments of the invention, analytes may be detectedand/or identified by any known method of Raman spectroscopy. In suchembodiments, the metal-coated porous substrate 110, 210 may be operablycoupled to one or more Raman detection units. Various methods fordetection of analytes by Raman spectroscopy are known in the art. (See,e.g., U.S. Pat. Nos. 6,002,471; 6,040,191; 6,149,868; 6,174,677;6,313,914). Variations on surface enhanced Raman spectroscopy (SERS),surface enhanced resonance Raman spectroscopy (SERRS), hyper-Ramanspectroscopy and coherent anti-Stokes Raman spectroscopy (CARS) havebeen disclosed. In SERS and SERRS, the sensitivity of the Ramandetection is enhanced by a factor of 10⁶ or more for molecules adsorbedon roughened metal 150 surfaces, such as silver, gold, platinum, copperor aluminum surfaces.

[0061] A non-limiting example of a Raman detection unit is disclosed inU.S. Pat. No. 6,002,471. An excitation beam may be generated by either afrequency doubled Nd:YAG laser 410 at 532 nm wavelength or a frequencydoubled Ti:sapphire laser 410 at 365 nm wavelength. Alternatively,excitation beams may be generated at 785 nm using a Ti:sapphire laser410 or 514 nm using an argon laser 410. Pulsed laser beams or continuouslaser beams may be used. The excitation beam passes through confocaloptics and a microscope objective, and is focused onto the Raman activesubstrate 110, 210 containing one or more analytes. The Raman emissionlight from the analytes is collected by the microscope objective and theconfocal optics and is coupled to a monochromator for spectraldissociation. The confocal optics includes a combination of dichroicfilters, barrier filters, confocal pinholes, lenses, and mirrors forreducing the background signal. Standard full field optics can be usedas well as confocal optics. The Raman emission signal is detected by aRaman detector 420, comprising an avalanche photodiode interfaced with acomputer for counting and digitization of the signal.

[0062] Another example of a Raman detection unit is disclosed in U.S.Pat. No. 5,306,403, including a Spex Model 1403 double-gratingspectrophotometer with a gallium-arsenide photomultiplier tube (RCAModel C31034 or Burle Industries Model C3103402) operated in thesingle-photon counting mode. The excitation source comprises a 514.5 mnline argon-ion laser 410 from SpectraPhysics, Model 166, and a 647.1 nmline of a krypton-ion laser 410 (Innova 70, Coherent).

[0063] Alternative excitation sources include a nitrogen laser 410(Laser Science Inc.) at 337 nm and a helium-cadmium laser 410 (Liconox)at 325 nm (U.S. Pat. No. 6,174,677), a light emitting diode 410, anNd:YLF laser 410, and/or various ion lasers 410 and/or dye lasers 410.The excitation beam may be spectrally purified with a bandpass filter(CHROMA) and may be focused on the Raman active substrate 110, 210 usinga 20× objective lens (Nikon). The objective lens may be used to bothexcite the analytes and to collect the Raman signal, by using aholographic beam splitter (Kaiser Optical Systems, Inc., Model HB647-26N18) to produce a right-angle geometry for the excitation beam andthe emitted Raman signal. A holographic notch filter (Kaiser OpticalSystems, Inc.) may be used to reduce Rayleigh scattered radiation.Alternative Raman detectors 420 include an ISA HR-320 spectrographequipped with a red-enhanced intensified charge-coupled device (RE-ICCD)detection system (Princeton Instruments). Other types of detectors 420may be used, such as Fourier-transform spectrographs (based onMichaelson interferometers), charged injection devices, photodiodearrays, InGaAs detectors, electron-multiplied CCD, intensified CCDand/or phototransistor arrays.

[0064] Any suitable form or configuration of Raman spectroscopy orrelated techniques known in the art may be used for detection ofanalytes, including but not limited to normal Raman scattering,resonance Raman scattering, surface enhanced Raman scattering, surfaceenhanced resonance Raman scattering, coherent anti-Stokes Ramanspectroscopy (CARS), stimulated Raman scattering, inverse Ramanspectroscopy, stimulated gain Raman spectroscopy, hyper-Ramanscattering, molecular optical laser examiner (MOLE) or Raman microprobeor Raman microscopy or confocal Raman microspectrometry,three-dimensional or scanning Raman, Raman saturation spectroscopy, timeresolved resonance Raman, Raman decoupling spectroscopy or UV-Ramanmicroscopy.

[0065] Raman Labels

[0066] Certain embodiments of the invention may involve attaching alabel to one or more analytes to facilitate their measurement by theRaman detection unit. Non-limiting examples of labels that could be usedfor Raman spectroscopy include TRIT (tetramethyl rhodamine isothiol),NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines,xanthines, succinylfluoresceins, aminoacridine, quantum dots, carbonnanotubes and fullerenes. These and other Raman labels may be obtainedfrom commercial sources (e.g., Molecular Probes, Eugene, Oreg.; SigmaAldrich Chemical Co., St. Louis, Mo.) and/or synthesized by methodsknown in the art.

[0067] Polycyclic aromatic compounds may function as Raman labels, as isknown in the art. Other labels that may be of use for particularembodiments of the invention include cyanide, thiol, chlorine, bromine,methyl, phosphorus and sulfur. The use of labels in Raman spectroscopyis known (e.g., U.S. Pat. Nos. 5,306,403 and 6,174,677). The skilledartisan will realize that the Raman labels used should generatedistinguishable Raman spectra and may be specifically bound to orassociated with different types of analytes.

[0068] Labels may be attached directly to the analytes or may beattached via various linker compounds. Cross-linking reagents and linkercompounds of use in the disclosed methods are known in the art. Ramanlabels that contain reactive groups designed to covalently react withother molecules, such as analytes, are commercially available (e.g.,Molecular Probes, Eugene, Oreg.). Methods for preparing labeled analytesare known (e.g., U.S. Pat. Nos. 4,962,037; 5,405,747; 6,136,543;6,210,896).

EXAMPLES Example 1 Construction of a Porous Silicon Substrate

[0069] Formation of Porous Nanocrystalline Silicon

[0070] Methods for making nanocrystalline porous silicon 110, 210 areknown in the art (e.g., U.S. Pat. No. 6,017,773). A layer ofnanocrystalline porous silicon 110, 210 may be formed electrochemicallyas disclosed in Petrova-Koch et al. (Appl. Phys. Let. 61:943, 1992).Depending on the particular application, the silicon may be lightly orheavily p-doped or n-doped prior to etching to regulate thecharacteristics of the porous silicon substrate 110, 210. Single crystalsilicon ingots may be grown by the well known Czochralski method (e.g.,http://www.msil.ab.psiweb.com/english/msilhist4-e.html). A singlecrystal silicon wafer may be treated with anodic etching in diluteHF/electrolyte to form a nanocrystalline porous silicon substrate 110,210. Alternatively, chemical etching in a solution of HF, nitric acidand water may be used without anodic etching. Ethanol may be used as awetting agent to improve pore wetting with the HF solution.

[0071] The wafer may be coated with polymethyl-methacrylate resist orany other known resist compound before etching. A pattern for thenanocrystalline porous silicon substrate 110, 210 may be formed bystandard photolithographic techniques. In different embodiments of theinvention, the nanocrystalline porous substrate 110, 210 may becircular, trench shaped, channel shaped or of any other selected shape.In certain embodiments, multiple porous substrates 110, 210 may beformed on a single silicon wafer to allow for multiple sampling channelsand/or chambers for Raman analysis. Each sampling channel and/or chambermay be operably coupled to one or more Raman detectors 420.

[0072] After resist coating and lithography, the wafer may be exposed toa solution of between about 15 to 50 weight percent HF in ethanol and/ordistilled water in an electrochemical cell comprised of Teflon®. Etchingmay be performed in the dark (p-type silicon) or in the light (n-type orp-type silicon). In different embodiments of the invention, the entireresist coated wafer may be immersed in an HF solution. In alternativeembodiments, the wafer may be held in place in the electrochemical cell,for example using a synthetic rubber washer, with only a portion of thewafer surface exposed to the HF solution (U.S. Pat. No. 6,322,895). Ineither case, the wafer may be electrically connected to the positivepole of a constant current source to form the anode of theelectrochemical cell. A platinum electrode may provide the cathode forthe cell. The wafer may be etched using an anodization current densityof between 5 to 250 milliamperes/cm² for between 5 seconds to 30 minutesin the dark, depending on the selected degree of porosity. In particularembodiments of the invention, a porosity of about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% may be selected. As isknown in the art, the anodization current density required to formporous silicon 110, 210 may depend in part on the type of siliconsubstrate used, such as whether the substrate is lightly or heavilyp-type (boron doped) or n-type (phosphorus doped)..

[0073] In other alternative embodiments of the invention, thenanocrystalline porous silicon substrate 110, 210 may be incorporatedinto a MEMS device comprising a variety of detectors 420, sensors,electrodes, other electrical components, mechanical actuators, etc.using known chip manufacturing techniques. In certain embodiments, suchmanufacturing procedures may occur before and/or after formation of theporous silicon substrate 110, 210 and/or coating with a Raman sensitivemetal 150.

Example 2 Metal Coating of Porous Silicon by Thermal Decomposition

[0074]FIG. 1 illustrates an exemplary method for uniformly impregnatingmetal 150 into nanoporous silicon 110. The surface of the porous silicon110 is oxidized to silicon dioxide 120 (FIG. 1B). A metal salt solution130 is diffused into the porous matrix 110 (FIG. 1C) and dried (FIG.1E). The dried metal salt 140 is thermally decomposed inside the poresto form a uniform metal layer 150 (FIG. 1F). Oxidation of the poroussilicon surface 110 enables complete wetting of porous silicon 110 inthe metal salt solution 130, while preventing spontaneous immersioncoating, which causes pore blockage. The dry metal salt 140 is thermallydecomposed in a furnace and pure metal 150 is deposited on the sidewalls of the nanopores. A uniform, thin metal 150 coating of nanoporoussilicon 110 may be obtained without plugging the pores, as oftenobserved with standard methods of metal 150 infiltration into nanoporoussilicon 110. Currently available plating methods are also diffusionlimited, resulting in non-uniform metal 150 deposition that can decreasethe reproducibility of analyte detection, depending upon where in themetal-coated substrate 110 the analyte is located.

[0075] An optimal immersion time and high metal ion concentration areneeded to make the metal 150 coat the entire porous structure 110. Theserequirements can be satisfied by oxidizing the surface of porous silicon110, either by chemical oxidation or plasma oxidation, prior to exposureto a metal salt solution 130 (FIG. 1B). Oxidation prevents spontaneousimmersion plating by stabilizing the porous surface 110. The oxidizedporous silion 110 may thus be immersed in highly concentrated metal saltsolution 130 without causing pore blockage (FIG. 1C). Excessive metalsalt solution 130 may be removed, for example by blowing nitrogen gas(FIG. 1D). The solvent is evaporated to increase absorption of metalsalt 140 on the porous surface 110 (FIG. 1E). The metal salts 140 arethermally decomposed (FIG. 1F) to form a uniform deposit of Raman activemetal 150 on the surface of the porous silicon substrate 110.

[0076] In a non-limiting example a porous silicon substrate 110 wasformed by electrochemical etching in a 15% HF solution, exposing borondoped crystalline silicon to a current density of 50 mA/cm². The poroussilicon substrate 110 was subjected to plasma oxidation in a Technicsoxygen plasma chamber at an oxygen flow rate of 50 sccm (standard cubiccentimeters) and radiofrequency power of 300 W (watts) for 20 min,resulting in formation of an approximately 50 Å (Angstrom) silicondioxide 120 layer on the surface of the pores. Alternatively, chemicaloxidation in piranha solution may be used (e.g.,http://www-device.eecs.Berkeley.edu/-daewon/labweek7.pdf). The silicondioxide 120 layer passivates the silicon dangling bond, preventing fastimmersion coating.

[0077] The oxidized porous silicon 110 was dipped in a 1 M AgNO₃solution 130 for 20 min at room temperature to completely wet the poreswith silver nitrate solution 130. Excessive silver nitrate solution 130was removed by nitrogen gun drying to prevent pore closure by excessivesilver 150 deposition. The solvent was removed from the remaining silvernitrate solution 130 by drying at 100° C. for 20 min. At this stage allthe solvent was evaporated and dry silver nitrate salt 140 was absorbedon the surface of pores, resulting in an observable brown color on thesurface of the porous silicon 110.

[0078] Thermal decomposition was performed for 30 min at 500° C. in anambient pressure furnace, resulting in the decomposition of the drysilver nitrate salt 140 to silver metal 150. The method disclosed hereinresulted in the formation of a highly uniform deposit of silver metal150 on the surface of the porous silicon substrate 110, as shown in FIG.5. FIG. 5 illustrates the silver depth profile obtained on nanoporoussilicon 110, as determined by Rutherford backscattering spectroscopyanalysis. The silver depth profile was compared for nanoporous silicon110 treated by conventional diffusion limited immersion plating in a 1mM AgNO₃ solution 130 for 2.5 min (FIG. 5A) versus the method of thepresent Example (FIG. 5B). As can be seen, the present method resultedin a highly uniform silver 150 deposit, of much greater penetrationdepth compared to the standard method (FIG. 5A and FIG. 5B). The presentmethod resulted in a uniform silver 150 deposit up to about 10 μm indepth (FIG. 5B), while the standard method resulted in a highlynon-uniform deposit of less than 3 μm in depth (FIG. 5A). The Rutherfordbackscattering data were corrected using scanning electron microscopyanalysis to determine the actual thickness of the porous silicon 110layer.

[0079] Comparing the distribution of silver 150 versus silicon using thepresent method (FIG. 5B), it is observed that the silver 150 depositionis uniform down to the level at which the silicon density reaches amaximum. That is, the data of FIG. 5B indicate that the metal deposit150 obtained with the present method extends homogeneously all the wayto the bottom of the pores in the porous silicon substrate 110. It isclear that using the standard method (FIG. 5A) the metal deposit 150ends well before the bottom of the pores.

Example 3 Raman Detection of Analytes

[0080] A Raman active metal-coated substrate 110, 210 formed asdisclosed above may be incorporated into a system 400 for Ramandetection, identification and/or quantification of analytes, asexemplified in FIG. 4. The substrate 110, 210 may be incorporated into,for example, a flow through cell, connected via inlet and outletchannels to one or more solvent reservoirs 220 and a waste reservoir230. Alternatively, the inlet channel may be connected to one or moreother devices, such as a sample injector and/or reaction chamber.Analytes may enter the flow through cell and pass across the Ramanactive substrate 110, 210, where they may be detected by a Ramandetection unit. The detection unit may comprise a Raman detector 420 anda light source 410, such as a laser. The laser 410 may emit anexcitation beam, activating the analytes and resulting in emission ofRaman signals. The Raman signals are detected by the detector 420. Incertain embodiments of the invention, the detector 420 may be operablycoupled to a computer that can process, analyze, store and/or transmitdata on analytes present in the sample.

[0081] In an exemplary embodiment of the invention, the excitation beamis generated by a titanium:sapphire laser 410 (Tsunami bySpectra-Physics) at a near-infrared wavelength (750-950 nm) or a galiumaluminum arsenide diode laser 410 (PI-ECL series by Process Instruments)at 785 nm or 830 nm. Pulsed laser beams or continuous beams may be used.The excitation beam is reflected by a dichroic mirror (holographic notchfilter by Kaiser Optical or an interference filter by Chroma or OmegaOptical) into a collinear geometry with the collected beam. Thereflected beam passes through a microscope objective (Nikon LU series),and is focused onto the Raman active substrate 110, 210 where targetanalytes are located. The Raman scattered light from the analytes iscollected by the same microscope objective, and passes the dichroicmirror to the Raman detector 420. The Raman detector 420 comprises afocusing lens, a spectrograph, and an array detector. The focusing lensfocuses the Raman scattered light through the entrance slit of thespectrograph. The spectrograph (RoperScientific) comprises a gratingthat disperses the light by its wavelength. The dispersed light isimaged onto an array detector (back-illuminated deep-depletion CCDcamera by RoperScientific). The array detector is connected to acontroller circuit, which is connected to a computer for data transferand control of the detector function.

[0082] In various embodiments of the invention, the detection unit iscapable of detecting, identifying and/or quantifying a wide variety ofanalytes with high sensitivity, down to single molecule detection and/oridentification. In certain embodiments of the invention, the analytesmay comprise single nucleotides that may or may not be Raman labeled. Inother embodiments, one or more oligonucleotide probes may or may not belabeled with distinguishable Raman labels and allowed to hybridize totarget nucleic acids in a sample. The presence of a target nucleic acidmay be indicated by hybridization with a complementary oligonucleotideprobe and Raman detection using the system 400 of FIG. 4. Alternatively,amino acids, peptides and/or proteins of interest may be detected and/oridentified using the disclosed methods and apparatus. The skilledartisan will realize that the methods and apparatus are not limiting asto the type of analytes that may be detected, identified and/orquantified, but rather that any analyte, whether labeled or unlabeled,that can be detected by Raman detection may be analyzed within the scopeof the claimed subject matter.

Example 4 Detection of Rhodamine 6G (R6G) by SERS

[0083]FIG. 6 illustrates the use of the disclosed methods, systems 400and apparatus for detection and identification of an exemplary analyte,rhodamine 6G (R6G) dye molecules. R6G is a well-characterized dyemolecule that may be obtained from standard commercial sources, such asMolecular Probes (Eugene, Oreg.). A 114 μM (micromolar) solution of R6Gwas prepared and analyzed by surface enhanced Raman spectroscopy (SERS),using a plasma-oxidized, dip and decomposed (PODD) silver-coated poroussilicon substrate 110 that was prepared by the method of Examples 1 and2. Porous silicon substrates 110 of varying degrees of average porositywere prepared by varying the etching conditions. The R6G solution wasdiffused into the PODD silver-coated substrate 110 and analyzed by SERS,according to the method of Example 3, using an excitation wavelength of785 nm. A chemical enhancer (lithium chloride or sodium bromide, about 1μM concentration) was added to enhance the Raman signal.

[0084] The resulting SERS emission spectra, obtained in PODDsilver-coated porous substrates 110 of varying porosity, are shown inFIG. 6. FIG. 6 shows SERS emission spectra for 114 μM R6G obtained ataverage porosities, in order from the lowest trace to the highest trace,of 52%, 55%, 65%, 70% and 77%. As indicated in FIG. 6, the intensity ofthe SERS emission peaks increases with increasing average porosity inthis range, with a highest intensity observed at 77% average porosity.Increasing the porosity above 77% pushes the porous silicon layer 110into a non-stable materials regime, which can result in physicalseparation of the porous layer 110 from the bulk silicon substrate. At77% porosity, scanning electron micrographs showed pore diameters ofabout 32 nm in width (not shown).

[0085] At 77% average porosity, a seven order of magnitude (10⁷)increase in intensity of the Raman emission spectrum was observed. Thiscompares with an approximately six order of magnitude enhancementobserved on a roughened silver 150 plate (not shown). Although theintensity of the SERS emission peaks increased as a function of averageporosity, the wavelengths of the emission peaks did not vary (FIG. 6),allowing the identification of R6G independent of the average porosityused. With an estimated detection volume of 1.25×10⁻¹⁶ liters, thecorresponding number of molecules of rhodamine 6 G detected wasapproximately 9 molecules.

[0086] Additional studies were performed with a solution of adenine,which is a more biologically relevant target molecule. Uniquespectroscopic features were detected from a 90 μM solution of adenine ona porous silicon substrate 110 coated with silver 150.

[0087] All of the METHODS, SYSTEMS 400 and APPARATUS disclosed andclaimed herein can be made and used without undue experimentation inlight of the present disclosure. It will be apparent to those of skillin the art that variations may be applied to the METHODS, SYSTEMS 400and APPARATUS described herein without departing from the concept,spirit and scope of the claimed subject matter. More specifically, itwill be apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of the claimedsubject matter.

What is claimed is:
 1. A method comprising: a) obtaining a poroussilicon substrate; b) oxidizing the surface of the substrate to formsilicon dioxide; c) wetting the substrate with a metal salt solution;and d) thermally decomposing the metal salt to form a metal deposit onthe substrate.
 2. The method of claim 1, further comprising removingexcess metal salt solution from the substrate.
 3. The method of claim 2,further comprising removing the solvent from the remaining metal saltsolution.
 4. The method of claim 3, wherein the solvent is removed bydrying at 100° C. for about 20 minutes.
 5. The method of claim 1,wherein the metal deposit comprises at least one Raman active metalselected from the group consisting of silver, gold, platinum, copper andaluminum.
 6. The method of claim 1, wherein the metal salt solutioncomprises silver nitrate.
 7. The method of claim 6, wherein the solutioncomprises between 0.5 molar and 5.0 molar silver nitrate.
 8. The methodof claim 1, wherein the porous silicon substrate has an average porosityof about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75% or 80%.
 9. The method of claim 1, wherein the porous siliconsubstrate is oxidized by plasma oxidation, chemical oxidation or thermalannealing.
 10. The method of claim 1, wherein the metal salt isthermally decomposed by heating to a temperature above 573° K.
 11. Amethod comprising: a) obtaining a porous membrane; b) inserting themembrane into a microfluidic pathway; and c) introducing a metal saltsolution into the membrane to form a metal deposit.
 12. The method ofclaim 11, wherein the membrane is a porous silicon membrane, a porouspolysilicon membrane, a porous aluminum membrane, a porous aluminamembrane or a porous metal grid.
 13. The method of claim 11, wherein themembrane is a porous silicon membrane.
 14. The method of claim 13,further comprising electropolishing the membrane.
 15. The method ofclaim 11, wherein the microfluidic pathway comprises one or more solventreservoirs.
 16. The method of claim 15, wherein two or more differentmetal salt solutions are introduced into the membrane.
 17. The method ofclaim 11, wherein the metal deposit comprises at least one Raman activemetal selected from the group consisting of silver, gold, platinum,copper and aluminum.
 18. A Raman detection system comprising ametal-coated porous substrate, wherein said substrate is produced by amethod comprising thermal decomposition or microfluidic impregnation ofa metal salt.
 19. The system of claim 18, wherein said substratecomprises at least one composition selected from the group consisting ofporous silicon, porous polysilicon, porous aluminum, porous alumina anda porous metal grid.
 20. The system of claim 18, wherein the substrateis a porous silicon substrate.
 21. The system of claim 18, furthercomprising a Raman detector and an excitatory light source.
 22. Thesystem of claim 21, further comprising a computer operably coupled tothe Raman detector.
 23. The system of claim 18, wherein the metal-coatedporous substrate is integrated into a microfluidic pathway.
 24. Thesystem of claim 23, wherein the pathway comprises one or more solventreservoirs.
 25. The system of claim 23, wherein the pathway comprises atleast one waste reservoir.
 26. The system of claim 18, furthercomprising an integrated microchip.
 27. The system of claim 18, whereinthe porous substrate is uniformly coated with at least one Raman activemetal.
 28. The system of claim 27, wherein the at least one Raman activemetal is selected from the group consisting of silver, gold, platinum,copper and aluminum.
 29. The system of claim 18, wherein the poroussubstrate has a metal penetration depth greater than 3 microns.
 30. Thesystem of claim 18, wherein the metal salt comprises silver nitrate orsilver sulfate.